Abstract
Alzheimer disease (AD) and chronic traumatic encephalopathy (CTE) involve the abnormal accumulation in the brain of filaments composed of both three-repeat (3R) and four-repeat (4R) (3R/4R) tau isoforms. To probe the molecular basis for AD’s tau filament propagation and to improve detection of tau aggregates as potential biomarkers, we have exploited the seeded polymerization growth mechanism of tau filaments to develop a highly selective and ultrasensitive cell-free tau seed amplification assay optimized for AD (AD real-time quaking-induced conversion or AD RT-QuIC). The reaction is based on the ability of AD tau aggregates to seed the formation of amyloid fibrils made of certain recombinant tau fragments. AD RT-QuIC detected seeding activity in AD (n = 16) brains at dilutions as extreme as 107–1010-fold, but was 102–106-fold less responsive when seeded with brain from most cases of other types of tauopathy with comparable loads of predominant 3R or 4R tau aggregates. For example, AD brains had average seeding activities that were orders of magnitude higher than Pick disease brains with predominant 3R tau deposits, but the opposite was true using our previously described Pick-optimized tau RT-QuIC assay. CTE brains (n = 2) had seed concentrations comparable to the weakest of the AD specimens, and higher than 3 of 4 specimens with 3R/4R primary age-related tauopathy. AD seeds shared properties with the tau filaments found in AD brains, as AD seeds were sarkosyl-insoluble, protease resistant, and reactive with tau antibodies. Moreover, AD RT-QuIC detected as little as 16 fg of pure synthetic tau fibrils. The distinctive seeding activity exhibited by AD and CTE tau filaments compared to other types of tauopathies in these seeded polymerization reactions provides a mechanistic basis for their consistent propagation as specific conformers in patients with 3R/4R tau diseases. Importantly, AD RT-QuIC also provides rapid ultrasensitive quantitation of 3R/4R tau-seeding activity as a biomarker.
Highlights
Alzheimer disease (AD) afflicts 5.7 million people in the US alone and their care is estimated to cost $232 billion annually
Using τ306 and previously described 3R tau RT-QuIC conditions [37], we saw more rapid seeding with 10−3 or 10−4 dilutions of AD brain homogenate relative to 10−3 dilutions of brain tissue that showed no tau pathology and was being used as control tissue harvested from individuals diagnosed as having Diffuse Lewy body Dementia (DLBD) (Online Resource Fig. 2a, c)
The addition of a threefold stoichiometric excess of a second tau-based substrate, K19CFh (Online Resource Fig. 2b, d), slowed the AD-seeded reactions, but allowed a more dilute AD homogenate (10−5) to be discriminated from homogenates prepared from brains that were found to be free of tau pathology in immunohistochemical analyses (Online Resource Fig. 2b, d)
Summary
Alzheimer disease (AD) afflicts 5.7 million people in the US alone and their care is estimated to cost $232 billion annually (http://www.Alz.org). The structures of the tau filaments of AD and Pick disease (PiD) have recently been shown to be distinct linear assemblies of tau molecules with parallel in-register intermolecular β-sheet amyloid architectures [12,13,14]. These are the first available structures of disease-associated tau aggregates, but they are unlikely to represent all pathological tau aggregates, because multiple permutations (referred to as strains) of tau aggregates have been isolated biologically and shown to propagate consistently in cell culture or in vivo [7, 23, 31]. The new NIA-AA Research Framework advocates the development of biological, biomarker-based, rather than primarily syndromal, definitions of AD, and their diagnostic and prognostic implications [22]
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